Growth hormone localization in the neural retina and retinal pigmented epithelium of embryonic chicks

It is well-established that growth hormone (GH) is present in the brain, spinal cord, and peripheral nerves of embryonic chicks, prior to the differentiation of pituitary somatotrophs, but its presence and distribution in retinal tissues is controversial. The possible presence of GH and GH mRNA in retinal tissues of early embryos has therefore been further evaluated. A 466-bp fragment of the pituitary GH cDNA, derived from a portion of exon 3 and spanning exons 4 and 5, was amplified by RT-PCR from reverse-transcribed mRNA from the pituitary glands of juvenile chicks and from the whole eye, neural retina, and retinal pigmented epithelium (RPE) of embryonic-day (ED) 9 chick embryos. In ED 9 embryos, GH immunoreactivity was demonstrated in the choroid and neural retina, in which it was particularly abundant in a layer of cells with the location and morphological appearance of retinal ganglion cells. GH immunoreactivity was also present in tissue sections of the RPE that were bleached to remove the melanin pigment. The intense GH staining in the RPE of ED 9 embryos was also revealed using a fluorescein-labeled GH antibody and confocal microscopy. At the ultrastructural level, GH detected by immunogold electron microscopy was present in the cytoplasm of RPE and neural retinal cells of ED 9 embryos. Although not associated with secretory granules, GH in the RPE was particularly associated with the membranes of the melanin granules. These results demonstrate that the neural retina and RPE are extrapituitary sites of GH production in early chick embryos, prior to the differentiation of the pituitary gland.     

Identification of horizontal cells generating different spectral responses in the retina of a teleost fish (Eugerres plumieri)

Six different types of spectral responses were recorded from horizontal cells under mesopic conditions in perfused retina, isolated from the dark-adapted mojarra (Eugerres plumieri). They were tentatively termed photopic Lr-, Lg1-, Lg2-, Lb-, and C-type, and scotopic L-type. The Lr-, Lg-, and Lb-type responses showed a maximum peak at 605, 550, and 516 nm respectively, while the C-type was composed of hyperpolarizing potentials in response to shorter wavelengths and depolarizing potentials in response to longer wavelengths (so-called R/G-type). The scotopic L-type has a peak at 516 nm in the spectral response and a slow decay phase in the waveform response. Following a brief period of diffuse illumination, it was found that the Lg1-type response is altered to the Lr-type, while both Lg2- and Lb-type responses change to the C-type. Intracellular marking with Lucifer or Procion yellow identified the cellular origins of different response types: external (He) and medial horizontal (Hm) cells for the Lr-type, internal horizontal (Hi) cells for the C-type, and rod-horizontal (Hr) cells for the scotopic L-type. Only He cells were found to possess an axon, while dye coupling was seen between axonless Hm, Hi, or Hr cells but not between He cells. The morphology of these fluorescent dye-marked cells was the same as that of the respective cells observed in Golgi-stained materials.     

Immunoreactive and biologically active somatostatin-like material in the human retina

[³HSpiroperidol and [³H](?)-sulpiride specific binding have been used to assay for D₁ and D₂ dopaminergic recognition sites in striatal membranes of aged rats. While [³H]spiroperidol binding shows a decreased number of binding sites, no changes have been detected in [³H](?)-sulpiride binding, which is a marker for D₂ dopaminergic receptors. Data obtained with GTP and DA-dependent adenylyl cyclase activity confirm the hypothesis that aging selectively affects in rats those dopaminergic receptors coupled to the formation of cyclic AMP (D₁).     

Drug-induced changes in catecholaminergic cells of the fish retina

The changes in fluorescence intensity and number of visible catecholaminergic cells (CA-cells), as revealed by means of a histofluorescence technique, were used as indicators of the effects of various pharmacological agents upon CA-cells in the retina of fishes (Cyprinus carpio and Eugerres plumieri). The study includes in vivo and in vitro experiments. In the in vivo experiments, intravitreal injection, two or three hours before eye enucleation, of 10 microgram L-DOPA, dopamine, or noradrenaline accentuated CA-cell fluorescence and increased the number of visible cells, whereas 10 microgram of tyramine, octopamine, synephrine, or adrenaline reduced the endogenous fluorescence. Intramuscular injection of reserpine (3 mg/kg) abolished CA-cell fluorescence. In the in vitro experiments, pieces of isolated retinas were incubated for three or 30 minutes in media containing different drugs. Only minor changes in fluorescence were detected after three minutes of incubation, but after 30 minutes, dopamine (20 microM) markedly enhanced CA-cell fluorescence. Carbachol (20 mM), acetylcholine (10 mM) plus BW-anticholinesterase (1 mM) or substance P(1.6 x 10(-2) mM), all reduced CA-cell fluorescence. Kainic acid (20 mM) abolished fluorescence from CA-cell somata, while fluorescent fiber networks remain unchanged. L-aspartate (5 mM) and GABA (10 mM) in the incubation medium did not influence fluorescence intensity. The results are relevant to, and consistent with, electrophysiological observations of dopamine-mediated spatial effects on horizontal cell potentials.     

Chromatic organization of retinal photoreceptors during eye migration of Atlantic halibut (Hippoglossus hippoglossus)

The retinas of fishes often have single and double cone photoreceptors that are organized in lattice-like mosaics. In flatfishes experiencing eye migration (i.e., the metamorphic process whereby one eye migrates to the other side of the head), the hexagonal lattice of single cones present in the larva undergoes major restructuring resulting in a dominant square mosaic postmetamorphosis consisting of four double cones surrounding each single cone. The expression of different opsin types during eye migration has not been examined despite its importance in understanding photoreceptor plasticity and whether cell fate (in terms of spectral phenotype) could influence square mosaic formation. Here, we probed the retina of Atlantic halibut undergoing eye migration for opsin expression using two antibodies, AHblue and AB5407, that labeled short wavelength sensitive 2 (SWS2) opsin and longer wavelength (predominantly middle wavelength sensitive, RH2) opsins, respectively. Throughout the retina, double and triple cones labeled with AB5407 exclusively, whereas the vast majority of single cones labeled with AHblue. A minority (<5%) of single cones in the square mosaic of the centroventral retina labeled with AB5407. In regions of mosaic transition and near peripheral growth zones, some single cones co-expressed at least two opsins as they labeled with both antibodies. Short wavelength (SWS2 expressing, or S) cones formed a nonrandom mosaic gradient from central to dorsal retina in a region dominated by the larval single cone mosaic. Our results demonstrate the expression of at least two opsins throughout the postmetamorphic retina and suggest opsin switching as a mechanism to create new cone spectral phenotypes. In addition, the S cone gradient at the onset of eye migration may underlie a plastic, cell induction mechanism by which a cone's phenotype determines that of its neighbors and the formation of the square mosaic.     

Small chicken growth hormone (scGH) variant in the neural retina

A novel variant of chicken growth hormone (cGH) that is severely truncated has recently been discovered in the neural retina. It is, however, unknown whether this protein binds to GH receptors (GHRs) and has biological activity. This possibility has therefore been addressed by homology modeling, using human (h)GH as a template because it is the only GH molecule with a crystal structure and because hGH binds to cGH receptors (cGHRs). Most of the residues of the small (s)cGH model fitted the hGH template, apart from two restricted regions from Ser 12 to Gln 20 and from Ser 55 to Val 58. The scGH model differs, however, from hGH in structure: hGH is composed of a four-helix bundle, whereas scGH has three main helices. Helices 2, 3, and 4 of hGH correspond to helices 1, 2, and 3 of scGH, but they are longer by one, four, and one residues, respectively. The secondary structure of the C-terminus of scGH is therefore similar to C-terminal hGH. The N-terminus of scGH is, however, severely truncated, lacking the residues of the full-length molecule derived from exons 1, 2, and 3. The N-terminus of scGH also includes 20 residues derived from intron C of full-length cGH. The predicted structure of its N-terminus has no classical secondary structure (alpha-helix or beta-sheet), whereas the N-terminus of hGH is composed of helix 1 and two mini-helices located between helix 1 and 2. This difference in ribbon structure results in a difference in the overall shape of the scGH model and hGH. The possibility that scGH could bind to a GHR dimer was assessed by examining the primary and hypothetical tertiary structure of scGH. hGH binds the extracellular domain (ECD) of two GHRs sequentially at its binding site 1 (or high affinity site) then at its binding site 2 (or low affinity site). Sequence alignment of scGH with hGH demonstrates that scGH lacks three key residues (of 14) at site 1 and nine residues (of 15) at site 2. It is therefore unlikely that tight binding of ECD1 to the site 1 of scGH could occur. scGH also lacks most of the site 2 residues, suggesting that it is unlikely that ECD2 would bind to the scGH model. In summary, we have developed a novel, structural model of scGH, with implications for its putative actions through classical GHRs.     

Identification and localization of myosin superfamily members in fish retina and retinal pigmented epithelium

Myosins are cytoskeletal motors critical for generating the forces necessary for establishing cell structure and mediating actin‐dependent cell motility. In each cell type a multitude of myosins are expressed, each myosin contributing to aspects of morphogenesis, transport, or motility occurring in that cell type. To examine the roles of myosins in individual retinal cell types, we first used polymerase chain reaction (PCR) screening to identify myosins expressed in retina and retinal pigmented epithelium (RPE), followed by immunohistochemistry to examine the cellular and subcellular localizations of seven of these expressed myosins. In the myosin PCR screen of cDNA from striped bass retina and striped bass RPE, we amplified 17 distinct myosins from eight myosin classes from retinal cDNA and 11 distinct myosins from seven myosin classes from RPE cDNA. By using antibodies specific for myosins IIA, IIB, IIIA, IIIB, VI, VIIA, and IXB, we examined the localization patterns of these myosins in retinas and RPE of fish, and in isolated inner/outer segment fragments of green sunfish photoreceptors. Each of the myosins exhibited unique expression patterns in fish retina. Individual cell types expressed multiple myosin family members, some of which colocalized within a particular cell type. Because much is known about the functions and properties of these myosins from studies in other systems, their cellular and subcellular localization patterns in the retina help us understand which roles they might play in the vertebrate retina and RPE. J. Comp. Neurol. 513:209–223, 2009. © 2009 Wiley‐Liss, Inc.     

Unique photoreceptor arrangements in a fish with polarized light discrimination

In contrast to other vertebrates, some anchovies have cone photoreceptors with longitudinally oriented outer segment lamellae. These photoreceptors are axially dichroic (i.e., they are sensitive to the polarization of axially incident light) and form the basis of a polarization detection system in the northern anchovy, Engraulis mordax. Whether other cone types exist in the retina of this animal, and whether multiple cone opsins are expressed in the retinas of anchovies, is unknown. Likewise, a detailed examination of photoreceptor ultrastructure in nondichroic photoreceptors has not been carried out despite its importance to understand visual specializations within the retina and its use in the formulation of models to explain cellular structure. Here, I combined light and electron microscopy with immunohistochemical studies of opsin expression to infer mechanisms of lamellar formation and to evaluate the potential for color vision in the northern anchovy retina. Morphological observations revealed three cone formations: 1) continuous rows made up of alternating long and short (bilobed) cones with longitudinally oriented lamellae that are orthogonal between cone types; 2) continuous rows of alternating long and short cones in which only the short cones have longitudinally oriented lamellae; and 3) rows of triple cones with transversely oriented lamellae, each triple cone consisting of two lateral cones flanking a small central cone. Ultrastructure investigations supported two models of outer segment formation resulting in the longitudinally oriented lamellae of long and short cones. In the case of the long cone, lateral compression of the outer segment, potentially via the formation of guanine platelet stacks in neighboring pigment epithelium cells, results in a shape transformation from conical to cunate and a tilt from transverse to longitudinal lamellae. In the case of the short (bilobed) cone, membrane invaginations from the connecting ciliary structure grow longitudinally to form a dichroic stack. Opsin expression studies indicated that all cones express middle-to-long wavelength opsins, with long and lateral cones possessing a different opsin from that in short and central cones, confirming the potential for color vision. Together with the ultrastructural observations, these results suggest that the unique cone topography in the northern anchovy retina may underlie a visual system with segregated color and polarization detection channels.     

Isolation and characterization of two teleost melanopsin genes and their differential expression within the inner retina and brain

Melanopsin is a newly discovered photopigment that is believed to be involved in the regulation of circadian rhythms in tetrapods. Here we describe the characterization of the first two teleost melanopsins (opn4a and opn4b) isolated from Atlantic cod (Gadus morhua). These two teleost genes belong to a subgroup of melanopsins that also include members from Xenopus, chicken, and Takifugu. In situ hybridization revealed that opn4a and opn4b are differentially expressed within the retina and brain. In the larval and adult retina, both melanopsins are expressed in a subset of cells in the inner retina, resembling amacrine and ganglion cells. In addition, opn4a is expressed in the horizontal cells, indicating a separate task for this gene. In the brain, the two melanopsins are separately expressed in two major retinal and extraretinal photosensitive integration centers, namely, the suprachiasmatic nucleus (opn4a) and the habenula (opn4b). The expression of opn4a in the suprachiasmatic nucleus in cod is similar to the melanopsin expression found in Xenopus. This suggests a conserved role for this opsin and an involvement in mediation of nonvisual photoreceptive tasks, such as entraining circadian rhythms and/or hypophysiotrophic systems. The differential expression of opn4b in the habenula suggests that this gene plays a role similar to that of opn4a, in that it is also situated in an area that integrates photic inputs from the pineal as well as other brain regions. Thus, the habenula may be an additional region that mediates photic cues in teleosts.     

Adaptive loss of ultraviolet-sensitive/violet-sensitive (UVS/VS) cone opsin in the blind mole rat (Spalax ehrenbergi)

In previous studies, fully functional rod and long-wavelength-sensitive (LWS) cone photopigments have been isolated from the eye of the subterranean blind mole rat (Spalax ehrenbergi superspecies). Spalax possesses subcutaneous atrophied eyes and lacks any ability to respond to visual images. By contrast this animal retains the ability to entrain circadian rhythms of locomotor behaviour to environmental light cues. As this is the only known function of the eye, the rod and LWS photopigments are thought to mediate this response. Most mammals are dichromats possessing, in addition to a single rod photopigment, two classes of cone photopigment, LWS and ultraviolet-sensitive/violet-sensitive (UVS/VS) with differing spectral sensitivities which mediate colour vision. In this paper we explore whether Spalax is a dichromat and has the potential to use colour discrimination for photoentrainment. Using immunocytochemistry and molecular approaches we demonstrate that Spalax is a LWS monochromat. Spalax lacks a functional UVS/VS cone photopigment due to the accumulation of several deleterious mutational changes that have rendered the gene nonfunctional. Using phylogenetic analysis we show that the loss of this class of photoreceptor is likely to have arisen from the visual ecology of this species, and is not an artefact of having an ancestor which lacked a functional UVS/VS cone photopigment. We conclude that colour discrimination is not a prerequisite for photoentrainment in this species.     

Genetic,temporal and developmental differences between melatonin rhythm generating systems in the teleost fish pineal organ and retina

Complete melatonin rhythm generating systems, including photodetector, circadian clock and melatonin synthesis machinery, are located within individual photoreceptor cells in two sites in Teleost fish: the pineal organ and retina. In both, light regulates daily variations in melatonin secretion by controlling the activity of arylalkylamine N-acetyltransferase (AANAT). However, in each species examined to date, marked differences exist between the two organs which may involve the genes encoding the photopigments, genes encoding AANAT, the times of day at which AANAT activity and melatonin production peak and the developmental schedule. We review the fish pineal and retinal melatonin rhythm generating systems and consider the evolutional pressures and other factors which led to these differences.     

Plasmalemmal and vesicular gamma-aminobutyric acid transporter expression in the developing mouse retina

Plasmalemmal and vesicular gamma-aminobutyric acid (GABA) transporters influence neurotransmission by regulating high-affinity GABA uptake and GABA release into the synaptic cleft and extracellular space. Postnatal expression of the plasmalemmal GABA transporter-1 (GAT-1), GAT-3, and the vesicular GABA/glycine transporter (VGAT) were evaluated in the developing mouse retina by using immunohistochemistry with affinity-purified antibodies. Weak transporter immunoreactivity was observed in the inner retina at postnatal day 0 (P0). GAT-1 immunostaining at P0 and at older ages was in amacrine and displaced amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), respectively, and in their processes in the inner plexiform layer (IPL). At P10, weak GAT-1 immunostaining was in Müller cell processes. GAT-3 immunostaining at P0 and older ages was in amacrine cells and their processes, as well as in Müller cells and their processes that extended radially across the retina. At P10, Müller cell somata were observed in the middle of the INL. VGAT immunostaining was present at P0 and older ages in amacrine cells in the INL as well as processes in the IPL. At P5, weak VGAT immunostaining was also observed in horizontal cell somata and processes. By P15, the GAT and VGAT immunostaining patterns appear similar to the adult immunostaining patterns; they reached adult levels by about P20. These findings demonstrate that GABA uptake and release are initially established in the inner retina during the first postnatal week and that these systems subsequently mature in the outer retina during the second postnatal week.     

Evidence That Corticotropin-Releasing Hormone Acts as a Growth Hormone-Releasing Factor in a Primitive Teleost, the European Eel (Anguilla anguilla)

The inhibitory control of growth hormone (GH) release by somatostatin (SRIH) has been conserved throughout vertebrate evolution. In contrast, the neuropeptides involved in the stimulatory control of GH vary according to species and/or physiological situations. We investigated the direct pituitary regulation of GH release in a primitive teleost, the European eel (Anguilla anguilla L.) at the juvenile stage. Short-term serum-free primary cultures of dispersed pituitary cells were used, and GH release was measured by an homologous radioimmunoassay. Whereas growth hormone-releasing hormone (GHRH), gonadotropin-releasing hormone (GnRH), thyrotropin-releasing hormone (TRH), neuropeptide Y (NPY) and cholecystokinin (CCK) failed to induce any change in GH release, corticotropin-releasing hormone (CRH) dose-dependently stimulated GH release with a significant effect at 1 nM and a maximal effect (> or =400% of controls at 24 h) at 100 nM. In agreement with our previous studies, PACAP also stimulated GH release but its maximal effect was lower than that of CRH. Proopiomelanocortin (POMC)-peptides, corticotropin (ACTH), melanotropin (alpha-MSH), beta-endorphin) had no effect on GH release, at any dose tested (0.1-1000 nM), indicating that the stimulatory effect of CRH on GH release by somatotrophs was not mediated by CRH-induced release of POMC-peptides from corticotrophs and melanotrophs. The CRH antagonist, alpha-helical CRH(9-41), significantly inhibited the stimulatory effect of CRH on GH release, suggesting the implication of specific CRH receptors related to mammalian ones. The stimulatory effect of CRH on GH release was reduced after 24 h of incubation, indicating a desensitization. In contrast, no desensitization to the inhibitory effect of SRIH was observed. SRIH inhibited CRH action in a dose-dependent manner. The effect of SRIH was overriding, 1 nM SRIH being able to abolish the effect of 1000 nM CRH. In conclusion, in the eel, CRH stimulates GH release directly at the pituitary cell level. GH and cortisol secretions could interact in controlling several physiological functions such as metabolism and ion exchange. This study suggests that CRH may have played an important early role in vertebrates co-ordinating the activation of various endocrine axes involved in metamorphosis, osmoregulation, stress and fasting. The stimulatory role of CRH on GH release may have been partially conserved during evolution, as it is found in some human physio-pathological situations such as stress, fasting and depression.     

Cone photoreceptor topography in the retina of sexually mature Pacific salmonid fishes

We examined the retinal cone topography in sexually mature individuals from four species of Pacific salmonid fishes by using semithin plastic sections. We identified variations in cone density and cone arrangements and noted the presence of putative ultraviolet (UV) cones. Putative UV cones were found over an area extending dorsotemporally from the center of the retina. Because most of the putative UV cones are believed to disappear in early ontogeny, their presence over a large proportion (15–20%) of the surface area of the adult retina suggests that they may be reincorporated prior to or at sexual maturity, at least in rainbow trout. Cone density varied across the retina, with highest values at the peripheral margin. Relatively high densities were observed ventrotemporally (in all specimens) and, to a lesser extent, dorsonasally (7 of 11 specimens). The higher cone density in the ventrotemporal retina may represent a retinal specialization in the part of the visual field located above and in front of the animal. Lowest cone densities were found dorsocentrally and coincided approximately with the distribution of putative UV cones, raising the possibility that these cones may not be used in visual tasks requiring the higher visual acuity normally associated with higher cone densities. We also report a novel cone arrangement that consists of rows of double cones inserted between rows composed of single–double cone pairs alternating in position. J. Comp. Neurol. 383:49–59, 1997. © 1997 Wiley-Liss, Inc.     

Anatomically specific changes in the expression of somatostatin, growth hormone-releasing hormone and growth hormone receptor mRNA in diabetic rats

Growth hormone (GH) secretion is altered in poorly controlled diabetic animals. However, modifications in the hypothalamic neuropeptides that control GH secretion, somatostatin and GH-releasing hormone (GHRH), as well as changes in the sensitivity of the hypothalamus and pituitary to the feedback effects of GH, are less clear. We have used RNase protection assays and in-situ hybridization to address whether the mRNA expression of GH, somatostatin and GHRH, as well as of the GH receptor (GHR) in the hypothalamus and anterior pituitary, are altered in streptozotocin-induced diabetic rats. After induction of diabetes, rats were treated with insulin twice daily for 3 weeks to obtain either poorly controlled (mean plasma glucose >300 mg/dl) or well-controlled diabetic rats. Although no significant change in pituitary GH mRNA expression was found, the hypothalamic expression of GHRH and somatostatin mRNA was reduced in poorly-controlled diabetic rats and returned to control values with normalisation of plasma glucose concentrations (P<0.0001 and P<0.002, respectively). Somatostatin mRNA expression was reduced only in the central portion of the periventricular nucleus, with no change being seen in the other areas of the periventricular nucleus or in the arcuate, suprachiasmatic or paraventricular nuclei. A significant decline in GHRH mRNA expression was observed in both the arcuate nucleus and ventromedial hypothalamus. Anterior pituitary GHR mRNA expression was significantly reduced in both well and poorly-controlled diabetic rats, while there was no change in the hypothalamus. To examine whether the evolution time of the diabetes influences these parameters, in a subsequent experiment, diabetic rats received no insulin for 2 months. A significant decline in GHRH and somatostatin mRNA expression was also observed in these rats. In addition, pituitary GH mRNA expression declined significantly in long-term diabetic rats. These results demonstrate that: (1) the expression of both GHRH and somatostatin declines specifically in anatomical areas involved in anterior pituitary hormone control; (2) GHR mRNA expression is decreased in the pituitary of diabetic rats, but not in the hypothalamus, and does not return to control values with normalisation of mean blood glucose concentrations; and (3) the evolution time of the diabetes is important for detecting some changes, including the decrease in pituitary GH mRNA expression.     

Retinal photoreceptor arrangement,SWS1 and LWS opsin sequence,and electroretinography in the South American marsupial Thylamys elegans (Waterhouse, 1839)

We studied the retinal photoreceptors in the mouse opossum Thylamys elegans, a nocturnal South American marsupial. A variety of photoreceptor properties and color vision capabilities have been documented in Australian marsupials, and we were interested to establish what similarities and differences this American marsupial showed. Thylamys opsin gene sequencing revealed two cone opsins, a longwave‐sensitive (LWS) opsin and a shortwave‐sensitive (SWS1) opsin with deduced peak sensitivities at 560 nm and 360 nm (ultraviolet), respectively. Immunocytochemistry located these opsins to separate cone populations, a majority of LWS cones (density range 1,600–5,600/mm²) and a minority of SWS1 cones (density range 100–690/mm²). With rod densities of 440,000–590,000/mm², the cones constituted 0.4–1.2% of the photoreceptors. This is a suitable adaptation to nocturnal vision. Cone densities peaked in a horizontally elongated region ventral to the optic nerve head. In ventral—but not dorsal—retina, roughly 40% of the LWS opsin‐expressing cones occurred as close pairs (double cones), and one member of each double cone contained a colorless oil droplet. The corneal electroretinogram (ERG) showed a high scotopic sensitivity with a rod peak sensitivity at 505 nm. At mesopic light levels, the spectral ERG revealed the contributions of a UV‐sensitive SWS1 cone mechanism and an LWS cone mechanism with peak sensitivities at 365 nm and 555 nm, respectively, confirming the tuning predictions from the cone opsin sequences. The two spectral cone types provide the basis for dichromatic color vision, or trichromacy if the rods contribute to color processing at mesopic light levels. J. Comp. Neurol. 518:1589–1602, 2010. © 2009 Wiley‐Liss, Inc.     

Visual pigments and opsin expression in the juveniles of three species of fish (rainbow trout,zebrafish, and killifish) following prolonged exposure to thyroid hormone or retinoic acid

Thyroid hormone (TH) and retinoic acid (RA) are powerful modulators of photoreceptor differentiation during vertebrate retinal development. In the embryos and young juveniles of salmonid fishes and rodents, TH induces switches in opsin expression within individual cones, a phenomenon that also occurs in adult rodents following prolonged (12 week) hypothyroidism. Whether changes in TH levels also modulate opsin expression in the differentiated retina of fish is unknown. Like TH, RA is essential for retinal development, but its role in inducing opsin switches, if any, has not been studied. Here we investigate the action of TH and RA on single‐cone opsin expression in juvenile rainbow trout, zebrafish, and killifish and on the absorbance of visual pigments in rainbow trout and zebrafish. Prolonged TH exposure increased the wavelength of maximum absorbance (λ_max) of the rod and the medium (M, green) and long (L, red) wavelength visual pigments in all fish species examined. However, unlike the opsin switch that occurred following TH exposure in the single cones of small juvenile rainbow trout (alevin), opsin expression in large juvenile rainbow trout (smolt), zebrafish, or killifish remained unchanged. RA did not induce any opsin switches or change the visual pigment absorbance of photoreceptors. Neither ligand altered cone photoreceptor densities. We conclude that RA has no effect on opsin expression or visual pigment properties in the differentiated retina of these fishes. In contrast, TH affected both single‐cone opsin expression and visual pigment absorbance in the rainbow trout alevin but only visual pigment absorbance in the smolt and in zebrafish. The latter results could be explained by a combination of opsin switches and chromophore shifts from vitamin A1 to vitamin A2. J. Comp. Neurol. 522:98–117, 2014. © 2013 Wiley Periodicals, Inc.     

Cyclic adenosine monophosphate as a second messenger in horizontal cell uncoupling in the teleost retina

The reduction in the receptive field of horizontal cells of the teleost Eugerres plumieri observed upon dopamine (DA) superfusion is thought to be due to cell uncoupling. The possible mechanisms by which activation of DA receptors modify the electric coupling between horizontal cells were studied in the present work. It was found that the effect of DA in different preparations is mediated by a modification of intracellular concentration of cAMP and H+. The effects of intracellular injection of cAMP and H+ were studied in retinal horizontal cells of the teleost E. plumieri. A triple microelectrode was used to inject the ion iontophoretically, to pass current pulses, and to record voltages from the same cell, while a fourth microelectrode was used to record voltages from a neighboring cell in the same retinal layer. Responses evoked by light spots and annuli were evaluated simultaneously. Coupling ratios between neighboring horizontal cells ranged from 0.22 to 0.45. The intercellular resistance (Rc), 0.5-3.5 x 10(6) ohms, and that of the remaining cell membrane resistance (Rm), 2.5-18 x 10(6) ohms, were calculated by means of a passive electrical model that has a hexagonal array. The microinjection of H+ with injection current from +5 to +30 nA for 40 to 100 sec led to temporary and reversible light response reduction. The coupling ratio between two impaled cells was reduced by about 30%, and intercellular resistance (Rc) increment was 320% while cell membrane resistance (Rm) did not change consistently. There was also a temporary and reversible Rm reduction (70-85%) and an Rc increment of 170-330% when cyclic adenosine monophosphate was iontophoretically injected with current from -30 to -40 nA for 50 to 170 sec. The coupling ratio between two impaled cells was reduced by about 40%, and light responses recorded from the injected cell showed a reduction in amplitude with the same time course as that of the resistive changes. The injection of Lucifer yellow into a horizontal cell under normal conditions always results in pronounced fluorescence for more distant cells; however, under constant injection of H+ or cAMP only the injected cell is fluorescent, which provides direct evidence of the reduction in the effectiveness of coupling between horizontal cells. The observed effects of intracellular H+ or cAMP injection correspond to the resistive changes in Rc and coupling ratio that occur in the horizontal cell network upon superfusion with a dopamine (DA) solution.